This PhD thesis focuses on Non-Homologous End Joining (NHEJ), one of the main DNA repair mechanisms in cells. NHEJ lies under the umbrella of the DNA Damage Response (DDR), a collection of cellular pathways that combat the tens of thousands DNA lesions a single cell experiences each day from both endogenous and exogenous sources (1). NHEJ, alongside other DNA repair pathways, is responsible for the repair of a subtype of DNA damage, defined as double-strand breaks (DSBs) (2, 3). Although less frequent than other types of damage, DSBs are the most perilous (3). If remained unrepaired or repaired incorrectly, they can be drivers for chromosomal rearrangements, cellular senescence, and apoptosis (2, 4). Despite its fundamental role in genomic stability, NHEJ has been implicated in carcinogenesis, tumour progression and radiotherapy and chemotherapy resistance (5). Since the 1980s when NHEJ was discovered, biochemical, cellular, and structural studies have led to a greater understanding of how the pathway progresses and the nature of the components involved. Briefly, DSBs are initially recognised by the Ku 70/80 (Ku) heterodimer (6), which subsequently recruits DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme complex, to mediate synapsis of the broken DNA ends (7, 8). Depending on the DNA end configuration, DNA end processing can follow by specialised processing enzymes, nucleases, and DNA polymerases to ensure ends are ready for ligation (9). Ligation is mediated by the DNA Ligase IV and X-ray cross-complementing protein 4 (XRCC4) complex (10), with the help of the XRCC4-like factor (XLF), which forms stabilising filaments with XRCC4 (11). Additional accessory factors also display scaffolding or enzymatic roles. However, the more we dive into NHEJ, the more we understand how highly complex this process is. NHEJ is very dynamic, and over the years, various scenarios of its assembly have been proposed. The proteins’ exact arrangement during pathway progression remains still largely unclear. Therefore, the aim of my work lies in further understanding the interactions that take place for efficient DSB repair by NHEJ. To achieve this, I have focused on further elucidating the interactome of the DSB recognition component, Ku. Ku bound to DSBs is regarded as the interaction hub of NHEJ (12-14), responsible for the recruitment of multiple NHEJ components to the site of damage. Deciphering the nature of these interactions should reveal how these components are arranged in space and time to mediate DSB repair but could also prove a fruitful avenue for specifically targeting NHEJ in a cancer setting. Despite strong biochemical and cellular evidence of these interactions, there is limited information on their structural arrangement. Benefiting from the “resolution revolution” of cryo-electron microscopy (cryo-EM) (15), during my PhD work, I aimed at elucidating the structural basis of the interactions of Ku with DNA and different NHEJ components. Interestingly, in the first cryo-EM models of Ku alone and bound to DNA and subsequent NHEJ complexes containing Ku, I was able to visualise the detailed molecular basis of its interaction with a previously identified but not fully examined small molecule stimulator of NHEJ, inositol hexakisphosphate (IP6) (16, 17), which co-purified alongside Ku. Following that, in collaboration with members of our group and cryo-EM facilities in Cambridge, the diverse interactome of Ku with NHEJ components such as DNA-PK, the Ligase IV-XRCC4 complex and XLF was visualised in the context of different multi-component assemblies, defined as NHEJ supercomplexes. Further attempts to obtain cryo-EM models of Ku with additional non-core NHEJ components were carried out, with some allowing the visualisation of high-resolution structures, such as that of Ku with the accessory factor Paralog of XRCC4 and XLF (PAXX) and other revealing the dimeric arrangement of NHEJ processing enzymes as DNA polymerase λ. Through collaborations with external academic and industrial partners, the importance of the identified interactions was further confirmed using biochemical, biophysical, and cellular studies. Beyond the NHEJ machinery, I aimed to examine the interactions of Ku with nucleosomes of different DNA lengths in an effort to move away from “naked” DNA and understand how NHEJ would assemble in a chromatin environment. Finally, given the implications of NHEJ in resistance to radiotherapy and chemotherapy, I aimed to develop a drug discovery pipeline to examine the druggability of protein-protein interactions (PPIs) in NHEJ, given their diversity and crucial role in pathway progression, as seen from our cryo-EM work. I focused on one interaction, that of Ku with XLF. Through in silico docking and subsequent experimental validation, I was able to identify two promising hits which could act as a basis for the development of inhibitors. In summary, the work presented in this PhD thesis describes the power provided by cryo-EM in allowing us to further elucidate the molecular basis of Ku’s interactome and its importance in pathway progression and a new avenue in exploring NHEJ PPIs as potential drug target.